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Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage

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Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage Molecular Pharmacology Fast Forward. Published on October 3, 2019 as DOI: 10.1124/mol.119.116657 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 116657 1. Title page Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage Clamp Fluorometry Lachlan Munro1, Lucy Kate Ladefoged2, Vithushan Padmanathan1, Signe Andersen1, Birgit Schiøtt2, 3, Anders S. Kristensen1 Downloaded from 1Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark molpharm.aspetjournals.org 2 Department of Chemistry, Aarhus University, Langelandsgade 140, DK-8000 Aarhus, Denmark 3Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Langelandsgade 140, DK- 8000 Aarhus, Denmark at ASPET Journals on October 1, 2021 1 Molecular Pharmacology Fast Forward. Published on October 3, 2019 as DOI: 10.1124/mol.119.116657 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 116657 2. Running title page Running title: Ligand Specific Conformational Changes at the 5-HT3 Receptor Corresponding author information: Anders S. Kristensen, Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. E-mail: [email protected]. Tel.: +45 35 30 Downloaded from 65 05 molpharm.aspetjournals.org Abbreviations used: 5-HT: 5-hydroxytryptamine; 5-HTBP: 5-HT binding protein; ACh: Acetylcholine; AChBP: Acetylcholine binding protein; CNS: central nervous system; Cryo-EM: Cryo-electron microscopy; DMEM: Dulbecco's modified Eagle's medium; ECD: extracellular domain; FPS: FRET positioning at ASPET Journals on October 1, 2021 and screening; GABA: γ-aminobutyric acid; h5-HT3A: human 5-HT3A; m5-HT3A: mouse 5-HT3A; mCPBG: 1-(m-chlorophenyl)-biguanide, MD: molecular dynamics; MTS-TAMRA: 2-((5(6)- tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate; pLGIC: pentameric ligand-gated ion channel; PMT: photomultiplier tube; TEVC: two-electrode voltage clamp; TMD: transmembrane domain; VCF: voltage-clamp fluorometry. 2 Molecular Pharmacology Fast Forward. Published on October 3, 2019 as DOI: 10.1124/mol.119.116657 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 116657 3. Abstract The 5-HT3 receptor is a member of the Cys-loop receptor super family of ligand-gated ion channel in the nervous system and is a clinical target in a range of diseases. The 5-HT3 receptor mediates fast serotonergic neurotransmission by undergoing a series of conformational changes initiated by ligand-binging that lead to the rapid opening of an intrinsic cation-selective channel. However, despite the availability of high-resolution structures of a mouse 5-HT3 receptor, many important aspects of the mechanistic basis of 5-HT3 receptor function and modulation by drugs Downloaded from remain poorly understood. In particular, there is little direct evidence for the specific conformational changes predicted to occur during ligand-gated channel activation and molpharm.aspetjournals.org desensitization. In the present study, we used voltage-clamp fluorometry (VCF) to measure conformational changes in regions surrounding the orthosteric binding site of the human 5-HT3A (h5-HT3A) receptor during binding of 5-HT and different classes of 5-HT3 receptor ligands. VCF utilizes parallel at ASPET Journals on October 1, 2021 measurements of receptor currents with photon emission from fluorescent reporter groups covalently attached to specific positions in the receptor structure. Reporter groups that are highly sensitive to the local molecular environment can in real-time report conformational changes as changes in fluorescence that can be correlated with changes in receptor currents reporting the functional states of the channel. Within the loop C-, D-, and E-regions that surround the orthosteric binding site in the h5-HT3A receptor, we identify positions that are amenable to tagging with an environmentally sensitive reporter group that reports robust fluorescence changes upon 5-HT binding and receptor activation. We use these reporter positions to characterize the effect of ligand binding on the local structure of the orthosteric binding site by agonists, competitive antagonists, and allosterically acting channel activators. We observed that loop C appears to show distinct fluorescence changes for ligands of the same class, while loop D reports similar fluorescence 3 Molecular Pharmacology Fast Forward. Published on October 3, 2019 as DOI: 10.1124/mol.119.116657 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 116657 changes for all ligands binding at the orthosteric site. In contrast, the loop E reporter position shows distinct changes for agonists, antagonists, and allosteric compounds, suggesting the conformational changes in this region are specific to ligand function. Interpretation of these results within the framework of current models of 5-HT3 and Cys-loop mechanisms are used to expand the understanding of how ligand binding in Cys-loop receptors relates to channel gating. Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2021 4 Molecular Pharmacology Fast Forward. Published on October 3, 2019 as DOI: 10.1124/mol.119.116657 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 116657 4. Significance statement The 5HT3 receptor is an important ligand-gated ion channel and drug target in the central and peripheral nervous system. Determining how ligand binding induced conformational changes in the receptor is central for understanding the structural mechanisms underlying 5HT3 receptor function. Here, we employ voltage-gated fluorometry to characterize conformational changes in the extracellular domain of the human 5HT3 receptor to identify intra-receptor motions during binding of a range of 5HT3 receptor agonists and antagonists. Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2021 5 Molecular Pharmacology Fast Forward. Published on October 3, 2019 as DOI: 10.1124/mol.119.116657 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 116657 5. Introduction 5-HT3 receptors in the central nervous system transduce fast synaptic transmission and are considered therapeutic targets for the treatment of chemotherapy-induced nausea and vomiting and psychiatric conditions such as schizophrenia and depression (Thompson & Lummis, 2007; Walstab et al., 2010). Furthermore, 5-HT3 receptors in the enteric nervous system regulate gut motility and are well-established targets for the treatment of irritable bowel syndrome (Lummis, 2012). 5-HT3 receptors belong to the pentameric ligand-gated ion channel (pLGIC) superfamily, also known as Downloaded from Cys-loop receptors (Nemecz et al., 2016). pLGICs have as structural hallmark five subunits forming a central ion-permeable pore in the transmembrane domain (TMD; Fig. 1A) with orthosteric ligand molpharm.aspetjournals.org binding sites located at subunit interfaces in the extracellular domain (ECD; Fig. 1A). pLGICs are thought to share a common structural mechanism for how agonist binding in the ECD couples to channel activation and desensitization in the TMD (Grosman et al., 2000; Barnes et al., 2009; Corringer et al., 2010; Lummis, 2012; Nys et al., 2013; Keramidas and Lynch, 2013; Nemecz et al., at ASPET Journals on October 1, 2021 2016). Detailed structural information is available for the 5-HT3 receptor in the form of X-ray crystal and cryo-electron microscopy (cryo-EM) structures of the mouse 5-HT3A (m5-HT3A) receptor (Hassaine et al., 2014; Basak, Gicheru, Rao, et al., 2018; Basak, Gicheru, Samanta, et al., 2018; Polovinkin et al., 2018). Also, the acetylcholine binding protein (AChBP), which is a soluble pentameric eukaryotic protein homologous to the ECD of Cys-loop receptors (Brejc et al., 2001; Sixma & Smit, 2003) has been engineered to mimic the 5-HT3 receptor ligand binding profile (Kesters et al., 2012). Crystal structures of this construct (5-HT binding protein, denoted 5-HTBP) in complex with 5-HT, granisetron (Kesters et al., 2012), palonosetron (Price et al., 2016) and varenicline (Price et al., 2015) provide atomic-level insight to potential ligand binding modes and local conformations of the ECD in the 5HT3 receptor. However, the binding mode of 5-HT, as well 6 Molecular Pharmacology Fast Forward. Published on October 3, 2019 as DOI: 10.1124/mol.119.116657 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 116657 as the local conformation of the binding sites observed in the 5-HTBP structure (Kesters et al., 2012), is different to the binding modes of 5-HT observed in recently reported cryo-EM structures of the mouse 5-HT3 (m5-HT3) receptor (Basak et al., 2018a; Polovinkin et al., 2018). Thus, there are still gaps in the understanding of how ligand binding in the orthosteric site can either induce or prevent the opening of the relatively distal ion channel; partly due to a lack of data describing specific intra-receptor motions during ligand binding. Voltage-clamp fluorometry (VCF) is a technique that uses site-specific labeling of proteins with Downloaded from an environmentally sensitive fluorophore coupled with two-electrode voltage-clamp electrophysiology (TEVC) (Mannuzzu et al., 1996).
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